Faculty of Agricultural Sciences Institute of Crop Science Section of Crop Physiology of Specialty

154 Pages·2015·5.95 MB·English

by  Michael Hagemann

Faculty of Agricultural Sciences
Institute of Crop Science
Section of Crop Physiology of Specialty Crops
University of Hohenheim

Prof. Dr. Jens Norbert Wünsche

Physiological and molecular mechanisms of fruitlet abscission in mango

Dissertation submitted in partial fulfilment of the requirements
for the degree of Doctor of Philosophy
in Agricultural Science

by
Michael Helmut Hagemann
University of Hohenheim

2015
Acknowledgements

I thank Prof. Dr. Wünsche for making this study possible and for his trust in me. His door
was always open for discussions and I highly appreciate his support on many different
levels. I am also grateful to Prof. Dr. Asch and to Prof. Dr. Weber for reviewing this thesis.

It always was a friendly and nice working environment in our wing of the castle and I
thank my colleagues for their valuable suggestions and plenty of technical help. Especially,
I thank Dr. Winterhagen and Dr. Hegele for their guidance and long discussions on the
topic and beyond. I also appreciate the support from the four B.Sc. students who partly
contributed to this thesis, especially M.Sc. Kofler and his committed field work.

I am also grateful for my friends from the Uplands Program. The PhD-time was sometimes
challenging but with mutual support we overcame all obstacles in Vietnam and in
Germany.

Special thanks to my loving and lovely family – the importance of a secure, inspiring and
always exciting childhood cannot be overrated and I am glad that this time is not about to
end. I thank my wife Sarah for her support and sacrifices. Thank you for your motivation,
encouragement and for your love.

This thesis was accepted as a doctoral dissertation in fulfillment of the requirement for the
degree of “Doktor der Agrarwissenschaften” (Dr. sc. agr.) at the Faculty of Agricultural
Science at the University of Hohenheim on 19 October 2015.

Date of oral examination: 19 October 2015

Examination committee

Vice-Dean and Head of the Examination Committee: Prof. Dr. H. Grethe

Supervisor and reviewer: Prof. Dr. J. N. Wünsche

Co-reviewer: Prof. Dr. F. Asch

Additional examiner: Prof. Dr. G. Weber

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Table of contents
Acknowledgements ............................................................................................................. II
Table of contents ............................................................................................................... III
List of figures ................................................................................................................... VII
List of tables .................................................................................................................... XIII
List of abbreviations ....................................................................................................... XIV
1. Introduction ................................................................................................................ 1
1.1 Mango .......................................................................................................................... 1
1.1.1 Origin and botany .................................................................................................................. 1
1.1.2 Production and cultivation practices ..................................................................................... 2
1.1.3 Specific features of the study area ......................................................................................... 3
1.2 General description of premature fruit drop in mango ................................................ 4
1.2.1 Patterns and intensity ............................................................................................................ 4
1.2.2 Possible causes and prevention ............................................................................................. 6
1.3 Mechanism of fruit abscission ..................................................................................... 9
1.3.1 Morphological changes ......................................................................................................... 9
1.3.2 Hormonal and molecular control........................................................................................... 9
1.4 Ethylene perception .................................................................................................... 14
1.5 Objectives and hypothesis .......................................................................................... 15
1.6 Publications ................................................................................................................ 16
2. A new approach for analyzing and interpreting data on fruit drop in mango .. 17
2.1 Abstract ...................................................................................................................... 17
2.2 Introduction ................................................................................................................ 18
2.3 Material and methods ................................................................................................. 21
2.3.1 Plant material and experimental sites. ................................................................................. 21
2.3.2 Environmental parameters. ................................................................................................. 21
2.3.3 Experimental design and treatments. .................................................................................. 22
2.3.4 Assessment of fruit drop. .................................................................................................... 23
2.3.5 Statistical analysis. .............................................................................................................. 24
2.4 Results ........................................................................................................................ 26
2.4.1 Assessment of fruit drop. .................................................................................................... 26
2.4.2 Cropping system evaluation. ............................................................................................... 29
2.4.3 Irrigation. ............................................................................................................................. 29
2.4.4 PGR application. ................................................................................................................. 30
2.5 Discussion .................................................................................................................. 31
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2.5.1 Fruit drop. ............................................................................................................................ 31
2.5.2 Climatic factors. .................................................................................................................. 34
2.5.3 Cropping system.................................................................................................................. 34
2.5.4 Irrigation. ............................................................................................................................. 34
2.5.5 PGR. .................................................................................................................................... 35
2.6 Conclusion ................................................................................................................. 36
2.7 Notes .......................................................................................................................... 36
2.8 Literature cited ........................................................................................................... 37
3. Ethephon induced abscission of mango fruitlets ̶ physiological fruit pedicel
response ..................................................................................................................... 41
3.1 Abstract ...................................................................................................................... 41
3.2 Introduction ................................................................................................................ 41
3.3 Material and methods ................................................................................................. 43
3.3.1 Plant material ...................................................................................................................... 43
3.3.2 Fruit and climate measurements .......................................................................................... 43
3.3.3 Leaf net carbon exchange rate ............................................................................................. 43
3.3.4 RNA extraction and gene expression analysis by quantitative real-time PCR ................... 43
3.3.5 Statistical analysis ............................................................................................................... 44
3.4 Results ........................................................................................................................ 45
3.4.1 Environmental conditions during natural fruit drop ............................................................ 45
3.4.2 Leaf net carbon exchange rate ............................................................................................. 46
3.4.3 Effectiveness of ethephon as fruit drop inducer .................................................................. 46
3.4.4 Ethylene receptors and their response to ethephon ............................................................. 47
3.4.5 Fruit detachment force ........................................................................................................ 48
3.5 Discussion .................................................................................................................. 49
3.6 Conclusion ................................................................................................................. 50
3.7 Acknowledgements .................................................................................................... 51
3.8 References .................................................................................................................. 51
4. Ethephon induced abscission in mango: physiological fruitlet responses .......... 53
4.1 Abstract ...................................................................................................................... 53
4.2 Introduction ................................................................................................................ 53
4.3 Materials and methods ............................................................................................... 56
4.3.1 Plant material and experimental site ................................................................................... 56
4.3.2 Treatments and experimental design ................................................................................... 56
4.3.3 Fruitlet drop assessment and sampling ................................................................................ 56
4.3.4 Gene analysis ...................................................................................................................... 57
4.3.5 Analysis of soluble carbohydrates ....................................................................................... 59
4.3.6 Polar auxin transport assay .................................................................................................. 60
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4.3.7 Statistical analysis ............................................................................................................... 60
4.4 Results ........................................................................................................................ 61
4.4.1 Ethephon induced fruitlet abscission ................................................................................... 61
4.4.2 Expression of ethylene receptors in the pedicel .................................................................. 62
4.4.3 Expression of ethylene receptors in the fruitlet pericarp ..................................................... 64
4.4.4 Polar auxin transport capacity ............................................................................................. 65
4.4.5 Analysis of soluble carbohydrates ....................................................................................... 66
4.5 Discussion .................................................................................................................. 67
4.6 Acknowledgement ..................................................................................................... 72
4.7 Supplementary material ............................................................................................. 72
4.7.1 Supplementary figures ........................................................................................................ 72
4.7.2 Supplementary tables .......................................................................................................... 77
4.8 References .................................................................................................................. 77
5. Expression and dimerisation of the mango ethylene receptor MiETR1 and
different receptor versions of MiERS1 .................................................................. 81
5.1 Abstract ...................................................................................................................... 81
5.2 Introduction ................................................................................................................ 81
5.3 Material and methods ................................................................................................. 83
5.3.1 Plant material ...................................................................................................................... 83
5.3.2 Sequence isolation and characterisation .............................................................................. 84
5.3.3 Gene expression studies by quantitative real-time PCR ..................................................... 84
5.3.4 Transient expression in Nicotiana benthamiana ................................................................. 85
5.4 Results and discussion ............................................................................................... 86
5.4.1 Sequence isolation and characterisation .............................................................................. 86
5.4.2 Presence of MiERS1 versions in different mango cultivars ................................................ 89
5.4.3 Expression analysis by quantitative real-time PCR ............................................................ 89
5.4.4 Mango ethylene receptor localisation in the plant cell ........................................................ 92
5.4.5 Co-localisation of mango ethylene receptors ...................................................................... 94
5.4.6 Dimerisation of MiERS1 and the shorter MiERS1 versions ............................................... 96
5.4.7 Dimerisation analysis by bi-molecular fluorescence complementation (BiFC) assay ........ 98
5.4.8 Summary and conclusion .................................................................................................... 99
5.5 Acknowledgements .................................................................................................. 100
5.6 Supplementary material ........................................................................................... 100
5.6.1 Supplementary tables ........................................................................................................ 101
5.6.2 Supplementary figures ...................................................................................................... 103
5.7 References ................................................................................................................ 107
6. General discussion .................................................................................................. 111
6.1 The molecular and physiological basis of fruitlet abscission .................................. 111
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6.2 Linking the theoretical understanding to practical solutions ................................... 116
6.3 Does it help to explain fruit drop patterns? .............................................................. 118
6.4 Recommendations for mango production ................................................................ 120
6.4.1 General recommendations ................................................................................................. 120
6.4.2 Recommendations specific for the study area ................................................................... 121
6.5 Conclusion and outlook ........................................................................................... 122
7. General references ................................................................................................. 123
8. Summary ................................................................................................................. 132
9. Zusammenfassung .................................................................................................. 134
10. Author’s declaration .............................................................................................. 136
11. Curriculum vitae .................................................................................................... 137

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List of figures
Fig. 1.1. Photograph series of the fruit of the cultivar ‘Hôi’. (A) ripe fruit, (B) fruit cut open
exhibiting the yellow mesocarp and seed, (C) seed and (D) embryo after removal of the
endocarp and testa. The several ventrodistally located embryos are characteristic for
polyembryonic cultivars (Arndt, 1935). ................................................................................... 4
Fig. 1.2. Different ways of presenting fruit drop in mango using the example of the cultivar
‘Tommy Atkins’. Graph was reproduced and modified based on Nuñez-Elisea and
Davenport (1983). Data are presented as (1) the decrease of fruited panicles (straight line,
black dot), (2) the decrease of fruit retention related to fruited panicles (dashed line, open
dot), (3) the decrease of fruit retention based on total initial panicles (straight line, open dot),
(4) and the rate of fruit drop per counting interval (dotted line, star). ..................................... 5
Fig. 1.3. Scheme of auxin flux in a fruit-bearing panicle of mango. The seed is considered as the
main source of auxin. Fruitlet 1: Seed degeneration leads to a decreased auxin efflux. This
enhances the sensitivity of the abscission zone (AZ) to ethylene (Roberts et al., 2002) and
can possibly also induce the ethylene biosynthesis (Abel et al., 1995), which subsequently
induces fruitlet abscission. Fruitlet 2: A fruitlet with a healthy seed is able to maintain a
sufficient auxin gradient that decreases the ethylene-sensitivity at the AZ, consequently the
fruit persists. Fruitlet 3: The strong auxin efflux from a further developed neighboring fruit
leads to the accumulation of auxin in the pedicel. This possibly triggers abscission either by
the auxin transport autoinhibition as proposed by Bangerth (1989) or through inducing the
ethylene biosynthesis (Abel et al., 1995). Fruitlet 4: The fruitlet has sufficient auxin efflux
and persists at the panicle. ..................................................................................................... 10
Fig. 1.4. Model for fruitlet abscission modified after Xie et al. (2011) considering Chen et al.
(2005) and Dal Cin et al. (2007, 2009). Ethylene is catalyzed from methionine by the
1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and the ACC oxidase
(ACO). The ACS activity can be modulated by auxin, gibberellins (GA), abscisic acid
(ABA), polyamines (PA) or others. In the absence of ethylene the ethylene receptors
maintain the CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) protein kinase in an active
state. The active CTR1 inhibits the ETHYLEN INSENSITIVE-2 (EIN2) signal protein.
Ethylene perception leads to a conformational change that inactivate CTR1, which in turn
induced the EIN2 transcription factors. EIN2 further induces EIN3 and EIN3-like (EIL),
which than induce the activity of transcription factors of the ETHYLENE RESPONSE
FACTOR (ERF) family in the nucleus. The ERFs lead to the upregulation of genes of the
ethylene signaling, which promotes the ethylene response, while the auxin signaling is
downregulated. In the following, the ERFs induce cell-wall degrading enzymes, which
finally leads to the detachment of the fruitlet and subsequently to the drop of the fruitlet. ... 13
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Fig. 2.1. (A) Average fruit retention per panicle (FPP) of ‘Hôi’ (closed symbols) and ‘Tròn’ (open
symbols) in days after full bloom (DAFB). Each symbol refers to years between 2007 and
2012 and is based on actual fruit counts per panicle. Average fruit retention over that period
is also modeled with a sigmoid function (r2 = 0.85). y is the final fruit retention, whereas
0
y +a determines the upper limit of FPP. (B) Slope of the simulated fruit drop curve and the
0
calculated daily rate of fruit drop FD(x). Black arrows indicate x , FD , and FD ,
0 max mst
corresponding with the highest daily fruit losses in absolute terms, the highest FD(x), and the
cessation of midseason fruit drop, respectively. .................................................................... 27
Fig. 2.2. Daily rate of fruit drop in six consecutive growing seasons. Gray lines indicate the fruit
drop waves by cultivar ‘Hôi’. ................................................................................................ 29
Fig. 3.1. Average maximum and minimum temperature during the flowering and early fruit
development period in 2010. Black dots indicate fruit counts of control treatments starting
one week after full bloom (7 February 2010). Error bars indicate SEM. .............................. 45
Fig. 3.2. Net carbon exchange rate (NCER) and stomatal conductance (SC) at four temperatures
during flowering in 2010. ...................................................................................................... 46
Fig. 3.3. Fruit retention during early fruit development in 2010 with application of Ethephon and
control treatments at an early (arrow 1) and a late application date (arrow 2). Error bars
represent SEM. ....................................................................................................................... 47
Fig. 3.4. Alignment of partial ETR1 and ERS1-like protein sequences with indicated homology
relative to the Arabidopsis sequence. ..................................................................................... 48
Fig. 3.5. Results of FDF measurements from the first experiment. Average FDF is shown at 2 days
before and 1, 3 and 5 days after application of Ethephon and control treatments. ................ 49
Fig. 4.1. The effect of the ethephon treatment 600 ppm (ET600) or 7200 ppm (ET7200) spray
applications on average (A) fruitlet retention, (B) fruitlet detachment force of fruitlets
detaching at the abscission zone or along the pedicel, (C) percentage of fruitlet detachment at
the abscission zone (the remainder to 100% are fruitlets detaching along the pedicel) and (D)
seed degeneration in comparison to the control and two days prior to treatment. (A)
Horizontal black bar indicates time until 95% of the fruits have abscised in response to
ET7200. (B) Homogeneous subgroups with no significant difference (p ≤ 0.05) are indicated
by same letters (a or b). Error bars show standard deviation. ................................................ 62
Fig. 4.2. Expression of the ethylene receptors (A) MiETR1 and (B) MiERS1 in the pedicel of pea
sized mango fruitlets in response to the ethephon treatment 600 ppm (ET600) and 7200 ppm
(ET7200) in comparison to the control and two days prior to treatment. Homogeneous
subgroups with no significant difference (p ≤ 0.05) are indicated by same letters (a,b or c).
Error bars show standard deviation. ....................................................................................... 63
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Fig. 4.3. Detection of transcription of the ethylene receptor versions (A) MiERS1m and
(B) MiERS1s in the pedicel of pea sized mango fruitlets in response to the ethephon
treatment 600 ppm (ET600) and 7200 ppm (ET7200) in comparison to the control and two
days prior to treatment. .......................................................................................................... 64
Fig. 4.4. Expression of the ethylene receptors (A) MiETR1and (B) MiERS1 in the pericarp of pea
sized mango fruitlets in response to the ethephon treatment 600 ppm (ET600) and 7200 ppm
(ET7200) in comparison to the control and two days prior to treatment. Homogeneous
subgroups with no significant difference (p ≤ 0.05) are indicated by same letters (a or b).
Error bars show standard deviation. ....................................................................................... 65
Fig. 4.5. Polar auxin transport (PAT) capacity through the pedicel of pea sized fruitlets. Detection
of [3H]-IAA in the receiver block in response to the ethephon treatment with 600 ppm
(ET600) or 7200 ppm (ET7200) in comparison to the control and two days prior to
treatment. Homogeneous subgroups with no significant difference (p ≤ 0.05) are indicated by
same letters (a or b). Error bars show standard deviation; dpm = disintegrations per minute.
1sample size (n=3) was too small to perform a statistical test. ............................................... 66
Fig. 4.6. Sucrose concentration of pea sized fruitlets after the ethephon treatment 600 ppm (ET600)
or 7200 ppm (ET7200) in comparison to the control and two days prior to treatment.
Homogeneous subgroups with no significant difference (p ≤ 0.05) are indicated by same
letters (a or b). Error bars show standard deviation. .............................................................. 67
Fig. 4.7. Overview of the key fruitlet abscission parameters analyzed in this study. Parameters of
ethephon treated fruitlets compared to those of control fruitlets: no significant differences are
indicated by a dot, whereas up- or downward pointing arrows indicate significant
differences. Different or equal response of the ethephon treatments 600 ppm (ET600) and
7200 ppm (ET7200) in comparison to the control are indicated by orange and cyan colored
symbols, respectively. The parameters are: fruitlet detachment force (FDF), gene expression
of the ethylene receptors MiERS1 and MiETR1 and their ratios in the pedicel and fruit
pericarp, polar auxin transport (PAT), and the concentration of sucrose in the fruit pericarp.
............................................................................................................................................... 71
Fig. 4.S1. The effect of ethephon treatment 7200 ppm (ET7200) on average (A) fruitlet detachment
force of fruitlets detaching at the abscission zone or along the pedicel and (B) percentage of
fruitlet detachment at the abscission zone (the remainder to 100% are fruitlets detaching
along the pedicel) in comparison to the control and one day prior to treatment. Error bars
show standard deviation. Data from 2011. ............................................................................ 72
Fig. 4.S2. Expression of the ethylene receptors (A) MiETR1 and (B) MiERS1 in the pedicel of pea
sized mango fruitlets in response to the ethephon treatment 7200 ppm (ET7200) in
comparison to the control and one day prior to treatment. Homogeneous subgroups with no
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significant difference (p ≤ 0.05) are indicated by same letters (a or b). Error bars show
standard deviation. Data from 2011. ...................................................................................... 73
Fig. 4.S3. Expression of short versions of the MiERS1. (A) MiERS1m and (B) MiERS1s in the
pedicel of pea sized mango fruitlets in response to the ethephon treatments 600 ppm (ET600)
or 7200 ppm (ET7200) in comparison to the control and two days prior to treatment. No
statistical test possible due to highly variable sample size. Missing error bar indicates n=1.
Data from 2012. ..................................................................................................................... 74
Fig. 4.S4. Expression analysis of (A) MiETR1 and (B) MiERS1 in the pericarp of pea sized fruitlets
in response to the ethephon treatment 7200 ppm (ET7200) in comparison to the control and
one day prior to treatment. Homogeneous subgroups with no significant difference (p ≤ 0.05)
are indicated by same letters (a or b). Error bars show standard deviation. Data from 2011. 75
Fig. 4.S5. Accumulated [3H]-IAA in the receiver after the ethephon treatment 7200 ppm (ET7200)
in comparison to the control and two days prior to treatment. Homogeneous subgroups with
no significant difference (p ≤ 0.05) are indicated by same letters (a or b). Error bars show
standard deviation; dpm = disintegrations per minute. Data from 2011. ............................... 76
Fig. 4.S6. Concentrations of fructose and glucose of pea sized fruitlets after the ethephon
treatments 600 ppm (ET600) or 7200 ppm (ET7200) in comparison to the control and two
days prior to treatment. Homogeneous subgroups with no significant difference (p ≤ 0.05)
are indicated by same letters (a or b). Error bars show standard deviation. Data from 2012. 76
Fig. 5.1. MiERS1 is expressed in three versions. (A) Structure and domains of the isolated MiERS1
versions. Domains are boxed in grey, broken line indicate gap in sequence, SNPs and their
positions are indicated. Numbers indicate position or sequence length in nucleotides (nt) or
amino acids (aa). TM: transmembrane domains, GAF: GAF domain, K: histidine kinase
domain, ATP: ATP-binding domain. (B) Alignment of translated cDNA sequences. Numbers
indicate sequence length in amino acids. (C) Genomic sequence structure of MiERS1
versions. Boxes indicate exons and thin lines indicate introns, numbers indicate size of
introns in nucleotides. ............................................................................................................ 87
Fig. 5.2. Expression analysis by qPCR of MiETR1 and the MiERS1 versions. Means of the level of
receptor transcripts in different mango organs and developmental stages relative to a linker
sample. MiETR1 (A) and MiERS1 (B) are present in 100% of the samples; occurrence of
MiERS1m (C) and MiERS1s (D) in all samples is indicated as percent in relation to tested
cDNAs. Expression values of MiERS1m and MiERS1s refer to samples with detectable
expression. Bars indicate standard deviation, except for (C) MiERS1m, marble pedicel due to
sample size n = 1. ................................................................................................................... 90
Fig. 5.3. Analysis of localisation of MiETR1 and the MiERS1 versions by transient expression in
infiltrated N. benthamiana leaves. Co-expression of MiETR1 (A) and MiERS1 version YFP-
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